WO2006133054A2 - Chemiluminescence proximity nucleic acid assay - Google Patents
Chemiluminescence proximity nucleic acid assay Download PDFInfo
- Publication number
- WO2006133054A2 WO2006133054A2 PCT/US2006/021675 US2006021675W WO2006133054A2 WO 2006133054 A2 WO2006133054 A2 WO 2006133054A2 US 2006021675 W US2006021675 W US 2006021675W WO 2006133054 A2 WO2006133054 A2 WO 2006133054A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- chemiluminescent
- molecule
- interest
- dye
- Prior art date
Links
- 238000007826 nucleic acid assay Methods 0.000 title description 2
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 161
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 157
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 157
- 238000000034 method Methods 0.000 claims abstract description 44
- 238000006243 chemical reaction Methods 0.000 claims description 59
- 238000009830 intercalation Methods 0.000 claims description 50
- 108060001084 Luciferase Proteins 0.000 claims description 47
- 239000005089 Luciferase Substances 0.000 claims description 39
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 claims description 38
- 230000000295 complement effect Effects 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 10
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 claims description 9
- 241001343649 Gaussia princeps (T. Scott, 1894) Species 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 6
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 claims description 4
- 241000963438 Gaussia <copepod> Species 0.000 claims description 4
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 claims description 3
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 claims description 3
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 241000254158 Lampyridae Species 0.000 claims description 2
- 241001443978 Oplophorus Species 0.000 claims 2
- 230000001580 bacterial effect Effects 0.000 claims 2
- AVNJFDTZJJNPKF-ZDUSSCGKSA-N 2-[3-[2-[(2S)-butan-2-yl]-3-hydroxy-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-8-yl]propyl]guanidine Chemical compound CC[C@H](C)c1nc2c(CCCNC(N)=[NH2+])nc(cn2c1[O-])-c1c[nH]c2ccccc12 AVNJFDTZJJNPKF-ZDUSSCGKSA-N 0.000 claims 1
- 241000006840 Acanthoptilum Species 0.000 claims 1
- 241000243290 Aequorea Species 0.000 claims 1
- 241000971686 Argyropelecus Species 0.000 claims 1
- 241001331164 Aristostomias Species 0.000 claims 1
- 241000881619 Chiroteuthis Species 0.000 claims 1
- 241000017626 Diaphus Species 0.000 claims 1
- 241000980703 Eucleoteuthis Species 0.000 claims 1
- 241001275863 Gnathophausia Species 0.000 claims 1
- 241000018620 Neoscopelus Species 0.000 claims 1
- 241001455233 Obelia Species 0.000 claims 1
- 241001533497 Odontosyllis Species 0.000 claims 1
- 241000882340 Onychoteuthis Species 0.000 claims 1
- 241000169268 Parazoanthus Species 0.000 claims 1
- 241001343647 Pleuromamma Species 0.000 claims 1
- 241000191235 Porichthys Species 0.000 claims 1
- 241001343656 Ptilosarcus Species 0.000 claims 1
- 241000242739 Renilla Species 0.000 claims 1
- 241000238371 Sepiidae Species 0.000 claims 1
- 241001002152 Sepiolina Species 0.000 claims 1
- 241000927894 Sergestes Species 0.000 claims 1
- 241000006838 Stylatula Species 0.000 claims 1
- 241000238430 Watasenia Species 0.000 claims 1
- 230000029918 bioluminescence Effects 0.000 claims 1
- 238000005415 bioluminescence Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- QUHVVVWAQMRCSJ-IXXPHHLHSA-N dinoflagellate luciferin Chemical compound N1C(CC2=C(C=3C(=O)CC(/C=3N2)=C/2[C@H]([C@H](C)[C@H](N\2)C(O)=O)CCC(O)=O)C)=C(CC)C(C)=C1CC1NC(=O)C(C)=C1C=C QUHVVVWAQMRCSJ-IXXPHHLHSA-N 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 30
- 239000000758 substrate Substances 0.000 abstract description 18
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 239000008241 heterogeneous mixture Substances 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 89
- 108020004414 DNA Proteins 0.000 description 87
- 239000000975 dye Substances 0.000 description 85
- 102000053602 DNA Human genes 0.000 description 48
- 239000003292 glue Substances 0.000 description 28
- 108010090804 Streptavidin Proteins 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 15
- 230000005284 excitation Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 238000002165 resonance energy transfer Methods 0.000 description 12
- 238000001228 spectrum Methods 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 238000000295 emission spectrum Methods 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- 238000009739 binding Methods 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 239000013615 primer Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- -1 deoxyribonucleotide triphosphates Chemical class 0.000 description 4
- 238000007429 general method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
- 229960005542 ethidium bromide Drugs 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Chemical group OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Chemical group OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000242764 Aequorea victoria Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- JBRZTFJDHDCESZ-UHFFFAOYSA-N AsGa Chemical compound [As]#[Ga] JBRZTFJDHDCESZ-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical group OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 108010076525 DNA Repair Enzymes Proteins 0.000 description 1
- 102000011724 DNA Repair Enzymes Human genes 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000009788 Exodeoxyribonucleases Human genes 0.000 description 1
- 108010009832 Exodeoxyribonucleases Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 229910001218 Gallium arsenide Inorganic materials 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000012330 Integrases Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000681013 Periphylla periphylla Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 101710086015 RNA ligase Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241001521365 Renilla muelleri Species 0.000 description 1
- 241000242743 Renilla reniformis Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229910052732 germanium Inorganic materials 0.000 description 1
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical compound [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
Definitions
- the detection system includes a chemiluminescent molecule, a chemiluminescent substrate, a dye that is light responsive when intercalated into nucleic acid and a nucleic acid target.
- the method requires that a specific three-dimensional structure (i.e. Dye intercalated into nucleic acid) be created for energy to be accepted by the dye and that the energy donor
- This invention is useful in any application where detection of a specific nucleic acid sequence is desirable, or where the detection of enzymes that modify nucleic acids is desirable such as diagnostics, research uses and industrial applications.
- Nucleic acids are measured to identify molecules of a specific target nucleic acid sequence in a population of heterogeneous nucleic acids, DNA or RNA, or to measure products of reactions where nucleic acids, DNA or RNA, are modified. Such measurements are generally permutations of the following procedures: a. where the starting nucleic acid is RNA, conversion to DNA is accomplished by a reverse transcription reaction. The oligonucleotide primers for the reverse transcription reaction may be specific for the target sequence or may be general for conversion of all RNA sequences to DNA; b. amplification of the target nucleic acid by target sequence specific reactions.
- PCR polymerase chain reaction
- primer extension reactions again with a target sequence specific oligonucleotide primer.
- Rolling circle amplification of DNA has also been used to amplify specific DNA sequences;
- physical separation of the heterogeneous nucleic acids include but are not limited to size fractionation and affinity separation when amplified nucleic acids are produced with derivatized substrates including but not limited to biotinylated deoxyribonucleotide triphosphates; d. labeling of the nucleic acid. As mentioned in c.
- amplified nucleic acids may be labeled using either derivatized deoxyribonucleotide triphosphates or derivatized oligonucleotide (KNA or DNA) primers; and e. detection of the nucleic acids. Nucleic acids can be detected either through the labeling moiety, or by physical separation followed by detection with nucleic acid specific dyes.
- KNA or DNA derivatized oligonucleotide
- One of the more common methods for the quantitative detection of target sequences is the sequence specific amplification of the target sequence(s) by PCR, either from DNA or from cDNA after reverse transcription, physical separation by gel or capillary electrophoresis, and detection by fluorescent labeling (e.g. of dsDNA by ethidium bromide or by use of fluorescently labeled primers in the amplification).
- Another common technique for the quantitative detection of target sequence(s) involves "real time" PCR.
- PCR technology is widely used to aid in quantitating DNA because the amplification of the target sequence allows for greater sensitivity of detection than could otherwise be achieved.
- the point at which the fluorescent signal is measured in order to calculate the initial template quantity can either be at the end of the reaction (endpoint QPCR) or while the amplification is still progressing (real-time QPCR).
- endpoint QPCR endpoint QPCR
- real-time QPCR real-time QPCR
- the reporter molecule used in real-time QPCR reactions can be (1) a sequence- specific probe composed of an oligonucleotide labeled with a fluorescent dye plus a quencher or (2) a non specific DNA binding dye that fluoresces when bound to double stranded DNA.
- a common method for the detection of nucleic acids is by staining them with fluorescing intercalating dyes.
- fluorescing intercalating dyes have several unique features that make them especially useful: 1) They have a high molar absorptivity; 2) Very low intrinsic fluorescence: 3) Large fluorescent enhancements upon binding to nucleic acids; and 4) Moderate to high affinity for nucleic acids, with little or no staining to other biopolymers.
- Intercalating nucleic acid stains have fluorescence excitations and emissions that span the visible-light spectrum from blue to near-infrared with additional absorption peaks in the UV, making them compatible with many different types of instrumentation. These dyes are excited with an extrinsic light source that has a spectrum that overlaps with the maximally excitation wavelength of the intercalated dye. They may be used to image both RNA and DNA. Some commonly used dyes are listed below.
- RET Resonance Energy Transfer
- FRET F ⁇ rster resonance energy transfer
- F ⁇ rster determined that the degree of resonance energy transfer between the energy donor and energy acceptor is inversely proportional to the distance between the two molecules to the sixth power.
- FRET an external light source of specific wavelength is used to excite the donor molecule.
- Bioluminescent Resonance Energy Transfer uses biological molecules such as a luciferase as the donor molecule. Depending on the species of origin, luciferases that use coelenterazine as a substrate generate blue light in the range of 450 to 500 nm. When a suitable acceptor is in close proximity, the blue light energy is captured by RET.
- the acceptor molecules are generally a class of proteins that have evolved the ability to be excited by blue light and then fluoresce in longer wavelengths typically with maximal spectral emissions above 500 nm.
- the molecules of interest may be either covalently or non-covalently linked or brought in to proximity by conformational change or by spatial migration or by an alteration in their relative orientations to one another.
- the two molecules may be conjugated to two separate proteins of interest. They may then be brought into proximity by their affinity for one another or their affinity for a third molecule. They may also be attached to a protein of interest and then brought closer due to a conformational change within the protein of interest.
- Luciferases that have been used in BRET include those from the firefly, Renilla reniformis and Gaussia princeps .
- a commonly used fluorescent protein is the green fluorescent protein (GFP) from Aequorea victoria.
- GFP green fluorescent protein
- BRET is generally used to measure the degree of affinity or degree of conformational change between two protein domains either covalently or non-covalently linked.
- the present invention functions to bring a Chemiluminescent Molecule within close proximity to dye stained target nucleic acids or the products of nucleic acid modifying reactions.
- the ability of the energy to be accepted by the dye is conditional. It is necessary for the dye to be intercalated into nucleic acid and that the energy donor be in close proximity.
- the method requires that a specific three-dimensional structure (i.e. Dye intercalated into nucleic acid that is contacted to a Chemiluminescent Molecule) be created for energy to be accepted by the dye and that the energy donor be proximal to this structure.
- the invention is rapid, and does not require any wash steps, which is significant as would be recognized by one of skill in the art. It does not require radioactivity nor does it require a laser for activating nucleic acid conjugated fluorophores.
- the signal from the emitted light in the reactions may be integrated over minutes as opposed to milliseconds as is the case with laser activated fluorophores.
- a unique aspect of this method is that of Proximity. Direct contact of the
- Chemiluminescent Molecule to the nucleic acid allows for the sensitive detection of a change in the mass of stainable nucleic acid (Example 3).
- the amount of fluorescence from nucleic acid that has been stained with an intercalating dye is directly proportional to the amount of nucleic acid present. Any condition in which the total mass of nucleic acid that is attached to the Chemiluminescent Molecule is increased or decreased will result in an increase or decrease of fluorescence by an activated intercalating dye.
- the Chemiluminescent Molecule does not simply act as an indicator of the presence of contact of a probe to a fluorophore.
- duplex nucleic acid is present by virtue of its illumination of dye bound that can only act as an energy acceptor when bound to duplex.
- detecting nucleic acids of specific sequence it adds a level of stringency.
- a positive signal requires both that the indicator molecule (i.e. the Chemiluminescent Molecule) be associated with the target sequence and also that nucleic acid be present.
- the indicator molecule i.e. the Chemiluminescent Molecule
- it demands that a specific three-dimensional structure be created for energy to be accepted by the dye and that the energy donor be a part and thus proximal to this structure. This will significantly reduce the background noise in the system for which it is being applied.
- the presence of the target nucleic acid is conveyed when the light emitting Chemiluminescent Molecule is brought into close proximity in the presence of fluorescent intercalated dye.
- the light emitted by the intercalated dye is proportional to the amount of stainable nucleic acid that is in close proximity to the Chemiluminescent Molecule.
- the present invention relates to a general detection system, for nucleic acids and methods of use thereof.
- the preferred system comprises four reagents: 1) a Chemiluminescent Molecule, 2) a Chemiluminescent Substrate, 3) an Intercalating Dye and 4) Nucleic Acid.
- These reagents are contacted with a Sample and can detect a change in the mass of stainable nucleic acid caused hybridization to complementary nucleic acids or by nucleic acid modifying reactions.
- the nucleic acids in a Sample can be either unamplified or the result of amplification reactions.
- a Chemiluminescent Probe may be made by covalently or non-covalently attaching the Chemiluminescent Molecule to a single stranded nucleic acid probe capable of hybridizing to complementary single stranded nucleic acid in the Sample.
- the target nucleic acid being probed in the Sample may be in solution phase with Chemiluminescent Probe in solution phase being added.
- the nucleic acid being probed in the Sample may be immobilized on a solid support with the Chemiluminescent Probe in solution phase being added.
- the Chemiluminescent Probe may be immobilized on a solid support with the nucleic acid being probed in the Sample in solution phase being added.
- the Chemiluminescent Probe and the nucleic acid being probed in the Sample may be immobilized.
- the Intercalating Dye is added to the Sample containing double stranded nucleic acid and a Chemiluminescent Molecule or Probe and it intercalates into the double stranded nucleic acid regions in the Sample.
- the Chemiluminescent Substrate is added to the Sample and is activated by the Chemiluminescent Molecule.
- the interaction of the Chemiluminescent Molecule and Chemiluminescent Substrate produces energy that in turn excites the Intercalating Dye at the Intercalating Dye Excitation Wavelength and the Intercalating Dye emits light at the Intercalating Dye Emission Wavelength.
- the light emitted at the Intercalating Dye Emission Wavelength is measured (with or without appropriate emission filters) and it is possible to determine the presence and quantitate the amount of target nucleic acid in the Sample.
- a filter one may discriminate longer wavelength light emitted by the fluorescing intercalated dye from the shorter wavelength light emitted by the Chemiluminescent Molecule. This discrimination may also be accomplished by incorporating into the Chemiluminescent Reaction non- intercalating, non- fluorescing dyes that absorb light emitted at the wavelengths produced by the Chemiluminescent Molecule but not that of the fluorescent intercalated dye. This general method is depicted in Figure 1.
- the Sample contains single stranded genomic DNA suspected of containing integrated HTV proviral sequence.
- the Chemiluminescent Molecule is Gaussia princeps luciferase (glue) and it is covalently attached to a ssDNA probe that is complementary to a region of the HIV envelope gene.
- the Intercalating Dye is PICOGREEN® and the Chemiluminescent Substrate is coelenterazine.
- the Sample DNA is denatured to generate single strands and then the probe covalently attached to Gaussia luciferase is added to the Sample and hybridizes to its complement.
- the PICOGREEN® is added to Sample and intercalates into the dsDNA region resulting from the probe hybridization.
- the coelenterazine is added to the Sample and causes the Gaussia luciferase to emit blue light with a spectrum peak at 480 nm.
- the emitted blue light causes any intercalated PICOGREEN® in close proximity to be excited, since its peak excitation wavelength is 502 nm.
- the PICOGREEN® then emits a bright green spectrum of light with a peak at 523 nm. that can be easily measured with a charged coupling device (CCD) camera that is equipped with a filter that significantly diminishes wavelengths below 500 nm.
- CCD charged coupling device
- An additional nonlimiting disclosure of the present invention would create a proximity assay by bringing a chemiluminescent molecule into close proximity with nucleic acid polymers incorporating fluorescently labeled nucleotides, or nucleotide analogs that fluoresce, in place of the intercalating dyes of the present invention.
- United States Patent Serial Nos. 6,451,536 and 6,960,436 describe the use of fluorescent nucleotides to detect and measure DNA samples without the component of proximity that embodies the present invention.
- This method is particularly well suited to detecting DNA in Samples either in solution or in a microarray format.
- This method is also well suited to detecting the products of enzymatic activities that create or modify nucleic acid samples such as polymerases, nucleases, recombinases and ligases as well detecting inhibitors of these activities.
- the present invention also encompasses methods of use of the above-described system.
- FIGURE 1 depicts an embodiment having Probe directly attached to
- Figure 2 depicts an embodiment having Probe directly attached to a Chemiluminescent Molecule and Sample is immobilized on Solid Support.
- Figure 3 depicts an embodiment having Probe indirectly attached to a Chemiluminescent Molecule and Sample both are unattached to a Solid Support.
- Figure 4 depicts an embodiment having Probe indirectly attached to a Chemiluminescent Molecule and Sample is immobilized on Solid Support.
- Figure 5A shows the CCD camera images for reactions described in Example 1 when SYBR GREEN I® is the Intercalating Dye.
- SYBR GREEN I® is the Intercalating Dye.
- the luciferase and SYBR GREEN I® concentrations are held constant while the DNA concentration is varied.
- Figure 5B shows the data generated in Example 1 presented as relative intensity per spot.
- Figure 6 A shows the CCD camera images for reactions described in Example 2 when SYBR GREEN I® is the Intercalating Dye.
- SYBR GREEN I® is the Intercalating Dye.
- the luciferase and DNA concentrations are held constant while the SYBR GREEN I® concentration is varied.
- Figure 6B shows the data generated in Example 1 presented as relative intensity per spot when SYBR GREEN I® is the Intercalating Dye.
- Figure 7A shows a schematic diagram of the experiment described in Example 3 where the biotinylated glue is brought into close proximity to the biotinylated DNA duplex by a streptavidin intermediate in the presence of SYBR GREEN I®.
- Figure 7B shows the data generated in Example 3 where the biotinylated glue is ⁇ brought into close proximity to the biotinylated DNA duplex by a streptavidin intermediate in the presence of SYBR Green I®.
- Figure 7B shows the data generated in Example 3 presented as relative intensity per spot when SYBR GREEN I® is the Intercalating Dye.
- Figure 8 shows the predicted data from a hypothetical assay using the Gaussia princeps luciferase conjugated to a DNA oligomer probe to quantitate DNA of a unique sequence in mixed sample.
- the invention provides a general method for detecting the presence or absence of nucleic acid in a Sample.
- the system comprises four reagents: 1) a Chemiluminescent Molecule, 2) a Chemiluminescent Substrate, 3) an Intercalating Dye and 4) Nucleic Acids.
- the following terms are intended to have the following general meanings as they are used herein as would be readily understood by one of skill in the art.
- Bioluminescent Molecule means any biological molecule involved in a chemiluminescent reaction.
- the reaction may be either catalytic or stoichiometric.
- “Chemiluminescent Emission Spectrum” means the range of photon wavelengths emitted by the Chemiluminescent Molecule. The spectrum is frequently defined by the wavelength of highest intensity from a chemiluminescent reaction.
- “Chemiluminescent Probe” means an olio- or poly- nucleotide probe molecule with a coupled Chemiluminescent Molecule. The Chemiluminescent Molecule may be coupled covalently or through non-covalent interaction, either before or after modification of the Probe by target nucleic acid.
- “Chemiluminescent Substrate” means a reactant that interacts with the
- Chemiluminescent Molecule to produce a photon/light.
- “Chemiluminescent Molecule” means any molecule that takes part in any chemiluminescent reaction; this includes but is not limited to a bioluminescent molecule.
- Various Chemiluminescent Molecules and their respective Chemiluminescent Substrates include but are not limited to: i) Luciferases that utilize coelenterazine as a Substrate including luciferases from the organisms Gaussia p ⁇ nceps, Periphylla periphylla, Renilla mulleri andAequorea Victori. ii) Firefly luciferase that utilizes firelfly luciferin as Substrate. iii) Alkaline phosphatase that utilizes DuoLuXTM Chemiluminescent/Fluorescent
- “Chemiluminescent Reaction” means any chemical reaction that produces a photon without an input photon.
- the reactants may act either catalytically or stoichiometrically.
- the catalyst converts a substrate(s) into a product(s) with the concomitant release of a photon.
- a stoichiometric reaction two or more reactants are converted to product(s) and a photon.
- “Complementary base pairs” means the purine and pyrimidine bases that pair to form stable hydrogen bonds between two single strand nucleic acid molecules.
- base pairs are adenine and thymidine, guanine and cytosine, and adenine and uracil.
- Other base pairs include derivatized variants of these bases, including but not limited to methylated bases, and other purines and pyrimidines including but not limited to inosine.
- "Double strand nucleic acid” means two single strand nucleic acid molecules that are non-covalently associated by hydrogen bonding of complementary bases on the two molecules.
- Excitation means the transfer of energy from a Chemiluminescent Molecule to the Intercalating Dye. Energy transfer from a luminescent molecule to the Intercalating Dye may be through the donation of photons or through Resonance Energy Transfer (RET).
- RET Resonance Energy Transfer
- Hybridization means the association reaction between two nucleic acid molecules through complementary base pairs to form a double strand nucleic acid.
- Intercalating Dye means a molecule that binds to double stranded or single stranded nucleic acids between adjacent base pairs. Further, upon intercalation the dye undergoes a change in its electronic configuration such that its absorption and/or emission spectra change. The dye has a very low intrinsic fluorescence when not bound to nucleic acids. The dye has a very large enhancement of fluorescence upon binding to nucleic acids with increases in quantum yields to as high as 0.9. The dye has a very high affinity for nucleic acids and little or no staining of other biopolymers.
- Intercalating Dye Excitation Spectrum means the range of wavelengths of energy that excites an intercalated dye complexed with double stranded or single stranded nucleic acid to produce a photon at its emission spectrum.
- the Intercalating Dye Excitation Spectrum overlaps with the emission spectrum of the Chemiluminescent Molecule.
- Intercalating Dye Emission Spectrum means the wavelengths of photons emitted by intercalated dye complexed with double stranded or single stranded nucleic acid when excited by a light source with a spectrum that overlaps with its maximal excitation wavelength.
- Nucleic acid means an oligomer or polymer of DNA, RNA or a chimera of both. It includes oligomers or polymers of DNA, RNA or chimeras of both into which analogs of nucleotides have been incoiporated. It also includes oligomers and polymers of nucleotide analogs, as would be recognized by one of skill in the ait.
- nucleotide analogs include nucleotides such as Locked Nucleic Acid (LNA) or Peptide Nucleic Acid (PNA) or other nucleotide analogs that are capable of complementary base- pairing with DNA or RNA, or nucleotide analogs that can be incorporated by enzymes that modify DNA such as telomerases, DNA polymerases, DNA repair enzymes, reverse transcriptases, or DNA and RNA ligases, or other DNA modifying enzymes known to those skilled in the art.
- “Probe” means any single strand nucleic acid with a defined sequence of purine and pyrimidine bases, including modifications as would be recognized by one of skill in the art.
- Proximity means the condition in which different molecules are close by virtue of their association in a stable molecular complex as would be appreciated by one of skill in the art.
- the molecules may be associated through covalent or non-covalent interactions. It is envisioned that the size of such complexes would be at the level seen in most protein/protein, protein/nucleic acid and nucleic acid/nucleic acid complexes.
- the proximity of the Chemiluminescent Molecule to nucleic acid would preferably be less than 500 A.
- the proximity of the Chemiluminescent Molecule to nucleic acid would more preferably be less than 250 A.
- the proximity of the Chemiluminescent Molecule to nucleic acid would most preferably be less than 100 A.
- the nucleic acid may have a length much greater than 500 A.
- Sample means any mixture of molecules collected from solid, solution or gas that may contain nucleic acid or activity that may modify nucleic acid or inhibitors of said activity.
- Single strand nucleic acid means an oligomer or polymer of repeating units of phosphate and ribose or deoxyribose joined at the 3 ' and 5 'positions of the sugar rings together with the purine or pyrimidine bases attached at the position of the ribose or deoxyribose ring.
- Solid support includes any suitable support for a binding reaction and/or any surface to which molecules may be attached through either covalent or non-covalent bonds.
- Probes may be attached to specific locations on the surface of a solid support in an addressable format to form an array, also referred to as a "microarray” or as a "biochip.”
- the preferred embodiment of the present invention comprises four molecules: the first is a Chemiluminescent Molecule, the second is a Chemiluminescent Substrate, the third is an Intercalating Dye and the fourth is Nucleic Acids.
- the absorption spectrum of the Intercalating Dye overlaps with the emission spectrum of the Chemiluminescent Molecule.
- the Chemiluminescent Molecule is linked, covalently or noncovalently, to a single strand nucleic acid complementary to the target sequence; this will be called the "Probe".
- the "Probe” nucleic acid is denatured and allowed to reanneal in the presence of the "Probe", the "Probe” and the target sequences in the Sample will form double stranded DNA.
- This double stranded DNA will in turn associate with the Intercalating Dye.
- the intercalation of the Intercalating Dye into double stranded will shift the absorption spectrum of the Intercalating Dye to overlap with the emission spectrum of the Chemiluminescent Molecule.
- the Chemiluminescent Molecule when provided with Chemiluminescent Substrate, it will generate the energy to excite the Intercalated Dye molecules and in turn cause them to emit photons at their emission wavelengths. These photons can be detected/counted.
- One method to quantitate the light emitted by the dye is to apply a filter that is able to discriminate between light emitted at the lower wavelength from light emitted by the intercalated dye. The efficiency with which energy produced by the Chemiluminescent Molecule is captured by the intercalated Intercalating Dye molecules will depend on the distance between them.
- the light emitted by the Intercalating Dye is a function the distance between the light source (Chemiluminescent Molecule) and the Intercalating Dye. If Excitation by the Chemiluminescent Molecule occurs by Resonance Energy Transfer then Forster's Equation applies. Forster's Equation states that the transfer of excitation energy between the donor (Chemiluminescent Molecule such as luciferase) and acceptor (Intercalating Dye such as PICOGREEN®) drops off as the 6 fll power of the distance between the two.
- An advantage of the present invention is that no light source aside from the Chemiluminescent Molecule is necessary for detection. Further, the association of the Chemiluminescent Probe with double strand DNA can be measured without physical separation of the target from other double strand nucleic acid, as only double strand DNA with intercalated Intercalating Dye by close physical association with the Chemiluminescent Probe will produce signal over background. This aspect of the invention alleviates the need for washes, a significant advantage as would be recognized by one with skill in the art. Any detector that can discriminate between the shorter and longer spectra wavelengths can be utilized in this assay system. These include, but are not limited to luminometers, fluorimeters, and CCD cameras equipped with a filter to remove shorter wavelengths in the range of that emitted by the Chemiluminescent Molecule.
- FIG 1 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence in solution phase.
- the Chemiluminescent Molecule is a luciferase.
- the luciferase is covalently attached to a ssDNA probe.
- This Bioluminescent Probe is added to a sample containing target sequence nucleic acid in solution and a nucleic acid stain.
- Coelenterazine is then added to activate the luciferase.
- the luciferase excites the nucleic acid intercalated stain that in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
- FIG. 2 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence immobilized on a solid support.
- the Chemiluminescent Molecule is a luciferase.
- the luciferase is covalently attached to a ssDNA probe.
- This Bioluminescent Probe is then added to the Sample with target nucleic acid immobilized in a solid support. Nucleic acid stain is present in solution in the
- luciferase excites the nucleic acid intercalated stain that in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
- FIG 3 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic of specific sequence in solution phase.
- the Chemiluminescent Molecule is a luciferase.
- the luciferase is noncovalently conjugated to a biotinylated ssDNA probe through a streptavidin intermediate. Because a single streptavidin molecule may bind four biotin molecules, biotinylated probe DNA, biotinylated luciferase and streptavidin may be mixed in the appropriate ratios to generate a Bioluminescent Probe.
- the Bioluminescent Probe is added to a sample containing target sequence nucleic acid and a nucleic acid stain.
- Coelenterazine is added to activate the luciferase.
- the luciferase then excites the nucleic acid intercalated stain, which in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
- FIG. 4 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence immobilized on a solid support.
- Chemiluminescent Molecule is a luciferase.
- the luciferase is noncovalently conjugated to a biotinylated ssDNA probe through a streptavidin intermediate. Because a single streptavidin molecule may bind four biotin molecules biotinylated probe DNA, biotinylated luciferase and streptavidin may be mixed in the appropriate ratios to generate a Bioluminescent Probe.
- This Bioluminescent Probe is then added to the Sample with target nucleic acid immobilized in a solid support. Nucleic acid stain is present in solution in the Sample. Coelenterazine is then added to activate the luciferase.
- the luciferase excites the nucleic acid intercalated stain, which in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
- the invention is a general method for detecting and quantitating target nucleic acid sequences in a heterogeneous mixture of nucleic acids.
- the detection of nucleic acids is important for many applications, including (but not limited to) diagnostic measurements of nucleic acids in bodily tissues and fluids as would be readily understood by one of skill in the art.
- the method serves to monitor the increase or decrease of stainable nuclei acid that is contacted to a Chemiluminescent Molecule.
- Stainable nucleic acid is any polymer of nucleic acid into which Intercalating Dyes will incorporate as opposed to other biological molecules. Upon binding, these Intercalating Dyes undergo a change in their electronic configuration that makes them fluoresce in the presence of the appropriate excitation wavelength.
- the method measures enzymatic activity that polymerizes the extension of a nascent strand, through the incorporation of nucleotides or nucleotide analogs, of nucleic acid when the extended strand or in the case of duplex its complement are contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present.
- Said activity includes but is not limited to RNA polymerases, DNA polymerases and telomerases.
- the invention also serves to detect inhibitors of the activities thereof;
- the method measures enzymatic activity that degrades nucleic acid when the nucleic acid is contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present.
- Said activity includes but is not limited to RNA exonucleases, RNA endonucleases, DNA exonucleases and DNA endonucleases.
- the invention also serves to detect inhibitors of the activities thereof;
- the method measures enzymatic activity that facilitates the attachment or ligation of separate nucleic acid molecules when one of the nucleic acid molecules is contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present. Said activity includes but is not limited to DNA ligases.
- the invention also serves to detect inhibitors of the activities thereof;
- the method measures enzymatic activity that facilitates the recombination of nucleic acid duplex molecules when one of the nucleic acid duplex molecules is contacted to the Chemiluminescent Molecule, a light responsive intercalating dye is present and the mass of the nucleic acid duplex of the recombined product is different than the mass of the nucleic acid duplex of the non-recombined molecule.
- Said activity includes but is not limited to recombinases and integrases.
- the invention also serves to detect inhibitors of the activities thereof;
- the method measures enzymatic activity that facilitates the attachment or ligation of duplex nucleic acid molecules to protein molecules when the protein molecules are contacted to or are a Chemiluminescent Molecule and a light responsive intercalating dye is present.
- the invention also serves to detect inhibitors of the activities thereof. All patents and publications referred to herein are expressly incorporated by reference in their entirety. The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
- the objective of this experiment was to determine if the luciferase enzyme of Gaussia princeps (glue) is sufficient to act as an excitation source for a fluorophore that is staining double stranded (ds) DNA.
- the experiment is intended to determine if the glue which emits light at apeak of 480 nm can excite a dsDNA intercalated nucleic acid stain with an excitation maximum in the range of 495 nm to 500 nm and an emission maximum of approximately 520 nm. This would be done by detecting light from a glue, fluorescing nucleic acid stain/ dsDNA mixture which has had wavelengths below 500 nm filtered out.
- the Gaussia princeps luciferase was from Avidity LLC (Denver, CO).
- the SYBR Green I nucleic acid stain and the linearized dsDNA ladder were obtained from Invitrogen (Carlsbad, CA).
- the reactions for the detection of dsDNA were as follows. Dilutions of dsDNA, SYBR Green I and glue were made with 50 mM Tris- HCl pH 7.8, 600 mM NaCl, 1 mM EDTA and 20% BPER II (Pierce Biotechnology, Rockford, IL). Coelenterazine (Nanolight International, Pinetop, CA) was diluted into PBS, 1 mM EDTA. The final concentration of coelenterazine in the reaction was 50 uM.
- dsDNA Various amounts of dsDNA were preincubated with 0.78 ⁇ l of 200 x concentrated SYBR Green I in a volume of 50 ⁇ l. A Ix concentration is relative and is that defined by the manufacturer as the standard assay amount for DNA detection.
- Glue (50ng) in 5 ⁇ l was added to the prestained dsDNA.
- the luciferase reaction was initiated by the addition of 100 ⁇ l of coelenterazine.
- the reactions were performed in the wells of a white polystyrene 96 multiwell plate (Evergreen Scientific, Los Angeles, CA). Light emitted by the reaction was detected with a CCD camera (Raytest, Straubenhardt, Germany).
- the filter (Clare Chemical Research, Dolores, CO) has been demonstrated to effectively image fluorescing nucleic acid stains that are excited by light wavelengths in the 400 nmto 500 nm range and emit at wavelengths higher than 500 nm when complexed with dsDNA. It does so by significantly filtering out wavelengths lower than 500 nm.
- Figure 5 depicts the light generating reaction performed with 50 ng of glue and coelenterazine in the presence of SYBR Green I and 0 ⁇ g, 0.063 ⁇ g, 0.125 ⁇ g, 0.25 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, and 2 ⁇ g of linearized dsDNA (spots 1-7 respectively).
- Part A shows the
- Example 2 Luciferase activation of a DNA intercalated dye is proportional to the amount of dye present.
- the objective of this experiment was to determine if the luciferase enzyme of Gaussia princeps (glue) is sufficient to act as an excitation source for a fluorophore that is staining double stranded (ds) DNA.
- the experiment is intended to determine if the glue which emits light at a peak of 480 nm can excite a dsDNA intercalated nucleic acid stain with an excitation maximum in the range of 495 nm to 500 nm and an emission maximum of approximately 520 nm. This would be done by detecting light from a glue, fluorescing nucleic acid stain/ dsDNA mixture which has had wavelengths below 500 nm filtered out.
- the amount of streptavidin increases the greater the number of molecules of dsDNA that are placed in close proximity to the glue molecules also increases. If the activation of the intercalated dye is dependent on the close proximity of the glue to the dye then the amount of fluorescence at longer wavelengths should increase as the amount of streptavidin increases.
- dsDNA used was made by annealing two complementary synthetic (Sigma-Genosys, The Woodlands, TX) oligonucleotides of DNA 85 and 95 nucleotides in length.
- One of the oligomers (95 nucleotides) was biotinylated at the 5' end.
- Streptavidin was from Pierce Biotechnology (Rockford, IL).
- dsDNA (2.5 pmole of biotinylated 5' end per reaction) was preincubated with
- Figure 7 depicts the light generating reaction performed with 50 ng of biotinylated glue and coelenterazine in the presence of PicoGreen dye.
- the DNA target for each reaction was at concentration of 2.5 pmole per biotinylated end per reaction.
- Streptavidin was present at 0.013 pmole, 0.026 pmole, 0.05 pmole, 0.1 pmole, 0.2 pmole, 0.42, 0.84 pmole (spots 1-8 respectively).
- Part A shows a schematic diagram of the experimental design.
- Part B shows the CCD camera images for the reactions with filter.
- Part C shows the CCD camera images for each reaction assessed as relative intensity per spot.
- the amount of light that can pass through the filter to the CCD camera is directly proportional to the amount of streptavidin that is added. All other components, luciferase, intercalating dye, dsDNA, and coelenterazine are the same in each reaction.
- the streptavidin serves to bring the Chemiluminescent Molecule (glue), and stained nucleic acid into a single complex in close proximity to one another. As more streptavidin is added more of the complex is created. The increase in complex is directly proportional the amount of longer wavelength light.
- Figure 8 depicts a hypothetical experiment representing a further application of the method.
- Each data point represents the intensity of a light emitting reaction with the amount of single stranded DNA target increasing in each reaction going from left to right.
- Gluc/DNA probe, PICOGREEN® and coelenterazine are held constant. Reactions are with either a) Probe with sequence complementary to the target DNA or b) Probe with sequence not complementary to the target DNA.
Abstract
This invention relates to the detection and quantitation of target nucleic acids in a heterogeneous mixture in a Sample and the methods of use thereof. The detection system includes a chemiluminescent molecule, a chemiluminescent substrate, a dye that is light responsive when intercalated into nucleic acids and nucleic acids. This invention is useful in any application where detection of a specific nucleic acid sequence is desirable, or where the detection of enzymes that modify nucleic acids is desirable such as diagnostics, research uses and industrial applications.
Description
CHEMELUMINESCENCE PROXIMITY NUCLEIC ACID ASSAY
FIELD OF THE INVENTION This invention relates to the detection and quantitation of target nucleic acids in a heterogeneous mixture in a Sample and the methods of use thereof. The detection system includes a chemiluminescent molecule, a chemiluminescent substrate, a dye that is light responsive when intercalated into nucleic acid and a nucleic acid target. The method requires that a specific three-dimensional structure (i.e. Dye intercalated into nucleic acid) be created for energy to be accepted by the dye and that the energy donor
(Chemiluminescent Molecule) be proximal to this structure. This invention is useful in any application where detection of a specific nucleic acid sequence is desirable, or where the detection of enzymes that modify nucleic acids is desirable such as diagnostics, research uses and industrial applications.
BACKGROUND OF THE INVENTION
Nucleic acids are measured to identify molecules of a specific target nucleic acid sequence in a population of heterogeneous nucleic acids, DNA or RNA, or to measure products of reactions where nucleic acids, DNA or RNA, are modified. Such measurements are generally permutations of the following procedures: a. where the starting nucleic acid is RNA, conversion to DNA is accomplished by a reverse transcription reaction. The oligonucleotide primers for the reverse transcription reaction may be specific for the target sequence or may be general for conversion of all RNA sequences to DNA; b. amplification of the target nucleic acid by target sequence specific reactions.
These include polymerase chain reaction (PCR) with sequence specific primers, and primer extension reactions again with a target sequence specific oligonucleotide primer. Rolling circle amplification of DNA has also been used to amplify specific DNA sequences; c. physical separation of the heterogeneous nucleic acids. Such physical separations include but are not limited to size fractionation and affinity separation when
amplified nucleic acids are produced with derivatized substrates including but not limited to biotinylated deoxyribonucleotide triphosphates; d. labeling of the nucleic acid. As mentioned in c. above, amplified nucleic acids may be labeled using either derivatized deoxyribonucleotide triphosphates or derivatized oligonucleotide (KNA or DNA) primers; and e. detection of the nucleic acids. Nucleic acids can be detected either through the labeling moiety, or by physical separation followed by detection with nucleic acid specific dyes.
One of the more common methods for the quantitative detection of target sequences is the sequence specific amplification of the target sequence(s) by PCR, either from DNA or from cDNA after reverse transcription, physical separation by gel or capillary electrophoresis, and detection by fluorescent labeling (e.g. of dsDNA by ethidium bromide or by use of fluorescently labeled primers in the amplification). Another common technique for the quantitative detection of target sequence(s) involves "real time" PCR.
PCR technology is widely used to aid in quantitating DNA because the amplification of the target sequence allows for greater sensitivity of detection than could otherwise be achieved. The point at which the fluorescent signal is measured in order to calculate the initial template quantity can either be at the end of the reaction (endpoint QPCR) or while the amplification is still progressing (real-time QPCR). The more sensitive and reproducible method of real-time QPCR measures the fluorescence at each cycle as the amplification progresses.
The reporter molecule used in real-time QPCR reactions can be (1) a sequence- specific probe composed of an oligonucleotide labeled with a fluorescent dye plus a quencher or (2) a non specific DNA binding dye that fluoresces when bound to double stranded DNA.
Both of these techniques, and others not described in detail, require instrumentation either for physical separation or detection. The requirement for instrumentation and/or separation technologies with their attendant sample handling limits the use of quantitative and qualitative target sequence detection. Accordingly, there is a need for methods of detecting and measuring nucleic acids that do not require
expensive, delicate instrumentation either for sample separation or for detection. Such measurements include but are not limited to the identification of molecules of a specific nucleic acid sequence as well as the detection of nucleic acids that are the product of nucleic acid modifying reactions. Nucleic acid modifying reactions include but are not limited to polymerization reactions, ligation reactions, nuclease reactions and recombination reactions.
Fluorescent Intercalating Nucleic Acid Dyes
A common method for the detection of nucleic acids is by staining them with fluorescing intercalating dyes. These dyes have several unique features that make them especially useful: 1) They have a high molar absorptivity; 2) Very low intrinsic fluorescence: 3) Large fluorescent enhancements upon binding to nucleic acids; and 4) Moderate to high affinity for nucleic acids, with little or no staining to other biopolymers.
Intercalating nucleic acid stains have fluorescence excitations and emissions that span the visible-light spectrum from blue to near-infrared with additional absorption peaks in the UV, making them compatible with many different types of instrumentation. These dyes are excited with an extrinsic light source that has a spectrum that overlaps with the maximally excitation wavelength of the intercalated dye. They may be used to image both RNA and DNA. Some commonly used dyes are listed below.
Dye Name Ex/Em * Application
Ethidium Bromide ; 300/600 \ Quantitation and Detection of dsDNA
I Ethidium Bromide 510/620 j Quantitation and Detection of dsDNA I Homodimer-1
PICOGREEN® 502/523 dsDNA Quantitation Reagent
OLIGREEN® ( 498/518 J Quantitation and Detection of ssDNA and Quantitation Reagent jj I oligonucleotides
of
Resonance Energy Transfer
Energy may be donated to nucleic acid intercalated dye either by photons or by resonance energy transfer. The principle of energy transfer between two molecules can be exploited as a means to provide information about relative changes in their proximity and orientation to one another. Resonance Energy Transfer (RET) is the transfer of excited state energy from a donor to an acceptor molecule. Fδrster resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. This can only occur if the absorption spectrum of acceptor molecule overlaps with the emission spectrum of the donor. Fδrster determined that the degree of resonance energy transfer between the energy donor and energy acceptor is inversely proportional to the distance between the two molecules to the sixth power. In the case of FRET, an external light source of specific wavelength is used to excite the donor molecule. Bioluminescent Resonance Energy Transfer (BRET) uses biological molecules such as a luciferase as the donor molecule. Depending on the species of origin, luciferases that use coelenterazine as a substrate generate blue light in the range of 450 to 500 nm. When a suitable acceptor is in close proximity, the blue light energy is captured by RET. The acceptor molecules are generally a class of proteins that have evolved the ability to be excited by blue light and then fluoresce in longer wavelengths typically with maximal spectral emissions above 500 nm. In both FRET and BRET the molecules of interest may be either covalently or non-covalently linked or brought in to proximity by conformational change or by spatial migration or by an alteration in their relative orientations to one another. For instance, the two molecules may be conjugated to two separate proteins of interest. They may then be brought into proximity by their affinity for one another or their affinity for a third molecule. They may also be attached to a protein of interest and then brought closer due to a conformational change within the protein of interest. Generally the two molecules must be within 100 A of one another for resonance energy transfer to occur and changes as little as 1-2 A may be detected. Luciferases that have been used in BRET include those from the firefly, Renilla reniformis and Gaussia princeps . A commonly used fluorescent protein is the green
fluorescent protein (GFP) from Aequorea victoria. BRET is generally used to measure the degree of affinity or degree of conformational change between two protein domains either covalently or non-covalently linked.
SUMMARY OF THE INVENTION
The present invention functions to bring a Chemiluminescent Molecule within close proximity to dye stained target nucleic acids or the products of nucleic acid modifying reactions. The ability of the energy to be accepted by the dye is conditional. It is necessary for the dye to be intercalated into nucleic acid and that the energy donor be in close proximity. The method requires that a specific three-dimensional structure (i.e. Dye intercalated into nucleic acid that is contacted to a Chemiluminescent Molecule) be created for energy to be accepted by the dye and that the energy donor be proximal to this structure.
Specific advantages of the present invention include the following. The invention is rapid, and does not require any wash steps, which is significant as would be recognized by one of skill in the art. It does not require radioactivity nor does it require a laser for activating nucleic acid conjugated fluorophores. The signal from the emitted light in the reactions may be integrated over minutes as opposed to milliseconds as is the case with laser activated fluorophores. A unique aspect of this method is that of Proximity. Direct contact of the
Chemiluminescent Molecule to the nucleic acid allows for the sensitive detection of a change in the mass of stainable nucleic acid (Example 3). The amount of fluorescence from nucleic acid that has been stained with an intercalating dye is directly proportional to the amount of nucleic acid present. Any condition in which the total mass of nucleic acid that is attached to the Chemiluminescent Molecule is increased or decreased will result in an increase or decrease of fluorescence by an activated intercalating dye. The Chemiluminescent Molecule does not simply act as an indicator of the presence of contact of a probe to a fluorophore. It indicates that duplex nucleic acid is present by virtue of its illumination of dye bound that can only act as an energy acceptor when bound to duplex. In the case of detecting nucleic acids of specific sequence it adds a level of stringency. A positive signal requires both that the indicator molecule (i.e. the
Chemiluminescent Molecule) be associated with the target sequence and also that nucleic acid be present. In other words it demands that a specific three-dimensional structure be created for energy to be accepted by the dye and that the energy donor be a part and thus proximal to this structure. This will significantly reduce the background noise in the system for which it is being applied.
The presence of the target nucleic acid is conveyed when the light emitting Chemiluminescent Molecule is brought into close proximity in the presence of fluorescent intercalated dye. The light emitted by the intercalated dye is proportional to the amount of stainable nucleic acid that is in close proximity to the Chemiluminescent Molecule.
The present invention relates to a general detection system, for nucleic acids and methods of use thereof. The preferred system comprises four reagents: 1) a Chemiluminescent Molecule, 2) a Chemiluminescent Substrate, 3) an Intercalating Dye and 4) Nucleic Acid. These reagents are contacted with a Sample and can detect a change in the mass of stainable nucleic acid caused hybridization to complementary nucleic acids or by nucleic acid modifying reactions. The nucleic acids in a Sample can be either unamplified or the result of amplification reactions.
A Chemiluminescent Probe may be made by covalently or non-covalently attaching the Chemiluminescent Molecule to a single stranded nucleic acid probe capable of hybridizing to complementary single stranded nucleic acid in the Sample. The target nucleic acid being probed in the Sample may be in solution phase with Chemiluminescent Probe in solution phase being added. The nucleic acid being probed in the Sample may be immobilized on a solid support with the Chemiluminescent Probe in solution phase being added. The Chemiluminescent Probe may be immobilized on a solid support with the nucleic acid being probed in the Sample in solution phase being added. The Chemiluminescent Probe and the nucleic acid being probed in the Sample may be immobilized.
The Intercalating Dye is added to the Sample containing double stranded nucleic acid and a Chemiluminescent Molecule or Probe and it intercalates into the double stranded nucleic acid regions in the Sample. The Chemiluminescent Substrate is added to the Sample and is activated by the Chemiluminescent Molecule. The interaction of the
Chemiluminescent Molecule and Chemiluminescent Substrate produces energy that in turn excites the Intercalating Dye at the Intercalating Dye Excitation Wavelength and the Intercalating Dye emits light at the Intercalating Dye Emission Wavelength. The light emitted at the Intercalating Dye Emission Wavelength is measured (with or without appropriate emission filters) and it is possible to determine the presence and quantitate the amount of target nucleic acid in the Sample. Using a filter one may discriminate longer wavelength light emitted by the fluorescing intercalated dye from the shorter wavelength light emitted by the Chemiluminescent Molecule. This discrimination may also be accomplished by incorporating into the Chemiluminescent Reaction non- intercalating, non- fluorescing dyes that absorb light emitted at the wavelengths produced by the Chemiluminescent Molecule but not that of the fluorescent intercalated dye. This general method is depicted in Figure 1.
In one non-limiting embodiment, the Sample contains single stranded genomic DNA suspected of containing integrated HTV proviral sequence. The Chemiluminescent Molecule is Gaussia princeps luciferase (glue) and it is covalently attached to a ssDNA probe that is complementary to a region of the HIV envelope gene. The Intercalating Dye is PICOGREEN® and the Chemiluminescent Substrate is coelenterazine. The Sample DNA is denatured to generate single strands and then the probe covalently attached to Gaussia luciferase is added to the Sample and hybridizes to its complement. The PICOGREEN® is added to Sample and intercalates into the dsDNA region resulting from the probe hybridization. The coelenterazine is added to the Sample and causes the Gaussia luciferase to emit blue light with a spectrum peak at 480 nm. The emitted blue light causes any intercalated PICOGREEN® in close proximity to be excited, since its peak excitation wavelength is 502 nm. The PICOGREEN® then emits a bright green spectrum of light with a peak at 523 nm. that can be easily measured with a charged coupling device (CCD) camera that is equipped with a filter that significantly diminishes wavelengths below 500 nm.
An additional nonlimiting disclosure of the present invention would create a proximity assay by bringing a chemiluminescent molecule into close proximity with nucleic acid polymers incorporating fluorescently labeled nucleotides, or nucleotide analogs that fluoresce, in place of the intercalating dyes of the present invention. United
States Patent Serial Nos. 6,451,536 and 6,960,436 describe the use of fluorescent nucleotides to detect and measure DNA samples without the component of proximity that embodies the present invention. These above referenced patents are hereby incorporated in their entirety by reference. This invention is useful in any application where detection of the presence or absence of DNA is desirable, such as diagnostics, research uses and industrial applications. This method is particularly well suited to detecting DNA in Samples either in solution or in a microarray format. This method is also well suited to detecting the products of enzymatic activities that create or modify nucleic acid samples such as polymerases, nucleases, recombinases and ligases as well detecting inhibitors of these activities. The present invention also encompasses methods of use of the above-described system.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts an embodiment having Probe directly attached to
Chemiluminescent Molecule and Sample where both are unattached to a Solid Support.
Figure 2 depicts an embodiment having Probe directly attached to a Chemiluminescent Molecule and Sample is immobilized on Solid Support.
Figure 3 depicts an embodiment having Probe indirectly attached to a Chemiluminescent Molecule and Sample both are unattached to a Solid Support.
Figure 4 depicts an embodiment having Probe indirectly attached to a Chemiluminescent Molecule and Sample is immobilized on Solid Support.
Figure 5A shows the CCD camera images for reactions described in Example 1 when SYBR GREEN I® is the Intercalating Dye. In this experiment the luciferase and SYBR GREEN I® concentrations are held constant while the DNA concentration is varied.
Figure 5B shows the data generated in Example 1 presented as relative intensity per spot.
Figure 6 A shows the CCD camera images for reactions described in Example 2 when SYBR GREEN I® is the Intercalating Dye. In this experiment the luciferase and
DNA concentrations are held constant while the SYBR GREEN I® concentration is varied.
Figure 6B shows the data generated in Example 1 presented as relative intensity per spot when SYBR GREEN I® is the Intercalating Dye. Figure 7A shows a schematic diagram of the experiment described in Example 3 where the biotinylated glue is brought into close proximity to the biotinylated DNA duplex by a streptavidin intermediate in the presence of SYBR GREEN I®.
Figure 7B shows the data generated in Example 3 where the biotinylated glue is ■ brought into close proximity to the biotinylated DNA duplex by a streptavidin intermediate in the presence of SYBR Green I®.
Figure 7B shows the data generated in Example 3 presented as relative intensity per spot when SYBR GREEN I® is the Intercalating Dye.
Figure 8 shows the predicted data from a hypothetical assay using the Gaussia princeps luciferase conjugated to a DNA oligomer probe to quantitate DNA of a unique sequence in mixed sample.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a general method for detecting the presence or absence of nucleic acid in a Sample. In a preferred embodiment, the system comprises four reagents: 1) a Chemiluminescent Molecule, 2) a Chemiluminescent Substrate, 3) an Intercalating Dye and 4) Nucleic Acids. The following terms are intended to have the following general meanings as they are used herein as would be readily understood by one of skill in the art.
A. Definitions
"Bioluminescent Molecule" means any biological molecule involved in a chemiluminescent reaction. The reaction may be either catalytic or stoichiometric.
"Chemiluminescent Emission Spectrum" means the range of photon wavelengths emitted by the Chemiluminescent Molecule. The spectrum is frequently defined by the wavelength of highest intensity from a chemiluminescent reaction.
"Chemiluminescent Probe" means an olio- or poly- nucleotide probe molecule with a coupled Chemiluminescent Molecule. The Chemiluminescent Molecule may be coupled covalently or through non-covalent interaction, either before or after modification of the Probe by target nucleic acid. "Chemiluminescent Substrate" means a reactant that interacts with the
Chemiluminescent Molecule to produce a photon/light.
"Chemiluminescent Molecule" means any molecule that takes part in any chemiluminescent reaction; this includes but is not limited to a bioluminescent molecule.
Various Chemiluminescent Molecules and their respective Chemiluminescent Substrates include but are not limited to: i) Luciferases that utilize coelenterazine as a Substrate including luciferases from the organisms Gaussia pήnceps, Periphylla periphylla, Renilla mulleri andAequorea Victori. ii) Firefly luciferase that utilizes firelfly luciferin as Substrate. iii) Alkaline phosphatase that utilizes DuoLuX™ Chemiluminescent/Fluorescent
Substrate for phosphatase, iv) Horseradish peroxidase that utilizes DuoLuX™
Chemiluminescent/Fluorescent Substrate for peroxidase
"Chemiluminescent Reaction" means any chemical reaction that produces a photon without an input photon. The reactants may act either catalytically or stoichiometrically. In the case of a catalytic reaction, the catalyst converts a substrate(s) into a product(s) with the concomitant release of a photon. In the case of a stoichiometric reaction, two or more reactants are converted to product(s) and a photon. "Complementary base pairs" means the purine and pyrimidine bases that pair to form stable hydrogen bonds between two single strand nucleic acid molecules. The usual base pairs are adenine and thymidine, guanine and cytosine, and adenine and uracil. Other base pairs include derivatized variants of these bases, including but not limited to methylated bases, and other purines and pyrimidines including but not limited to inosine.
"Double strand nucleic acid" means two single strand nucleic acid molecules that are non-covalently associated by hydrogen bonding of complementary bases on the two molecules.
"Excitation" means the transfer of energy from a Chemiluminescent Molecule to the Intercalating Dye. Energy transfer from a luminescent molecule to the Intercalating Dye may be through the donation of photons or through Resonance Energy Transfer (RET).
"Hybridization" means the association reaction between two nucleic acid molecules through complementary base pairs to form a double strand nucleic acid. "Intercalating Dye" means a molecule that binds to double stranded or single stranded nucleic acids between adjacent base pairs. Further, upon intercalation the dye undergoes a change in its electronic configuration such that its absorption and/or emission spectra change. The dye has a very low intrinsic fluorescence when not bound to nucleic acids. The dye has a very large enhancement of fluorescence upon binding to nucleic acids with increases in quantum yields to as high as 0.9. The dye has a very high affinity for nucleic acids and little or no staining of other biopolymers.
"Intercalating Dye Excitation Spectrum" means the range of wavelengths of energy that excites an intercalated dye complexed with double stranded or single stranded nucleic acid to produce a photon at its emission spectrum. The Intercalating Dye Excitation Spectrum overlaps with the emission spectrum of the Chemiluminescent Molecule.
"Intercalating Dye Emission Spectrum" means the wavelengths of photons emitted by intercalated dye complexed with double stranded or single stranded nucleic acid when excited by a light source with a spectrum that overlaps with its maximal excitation wavelength.
"Nucleic acid" means an oligomer or polymer of DNA, RNA or a chimera of both. It includes oligomers or polymers of DNA, RNA or chimeras of both into which analogs of nucleotides have been incoiporated. It also includes oligomers and polymers of nucleotide analogs, as would be recognized by one of skill in the ait. Examples of nucleotide analogs include nucleotides such as Locked Nucleic Acid (LNA) or Peptide Nucleic Acid (PNA) or other nucleotide analogs that are capable of complementary base-
pairing with DNA or RNA, or nucleotide analogs that can be incorporated by enzymes that modify DNA such as telomerases, DNA polymerases, DNA repair enzymes, reverse transcriptases, or DNA and RNA ligases, or other DNA modifying enzymes known to those skilled in the art. "Probe" means any single strand nucleic acid with a defined sequence of purine and pyrimidine bases, including modifications as would be recognized by one of skill in the art.
"Proximity" means the condition in which different molecules are close by virtue of their association in a stable molecular complex as would be appreciated by one of skill in the art. The molecules may be associated through covalent or non-covalent interactions. It is envisioned that the size of such complexes would be at the level seen in most protein/protein, protein/nucleic acid and nucleic acid/nucleic acid complexes. The proximity of the Chemiluminescent Molecule to nucleic acid would preferably be less than 500 A. The proximity of the Chemiluminescent Molecule to nucleic acid would more preferably be less than 250 A. The proximity of the Chemiluminescent Molecule to nucleic acid would most preferably be less than 100 A. The nucleic acid may have a length much greater than 500 A.
"Sample" means any mixture of molecules collected from solid, solution or gas that may contain nucleic acid or activity that may modify nucleic acid or inhibitors of said activity.
"Single strand nucleic acid" means an oligomer or polymer of repeating units of phosphate and ribose or deoxyribose joined at the 3 ' and 5 'positions of the sugar rings together with the purine or pyrimidine bases attached at the position of the ribose or deoxyribose ring. "Solid support" includes any suitable support for a binding reaction and/or any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, plastics, paramagnetic beads, charged paper, nylon, Langmuir-Blodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold and silver. Any other material known in the ait that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any
topology, including, but not limited to, flat surfaces, spherical surfaces, grooved surfaces, and cylindrical surfaces e.g., columns. Probes may be attached to specific locations on the surface of a solid support in an addressable format to form an array, also referred to as a "microarray" or as a "biochip."
B. The General Method (the illustrative embodiments are not exhaustive of the embodiments disclosed in the present invention)
The preferred embodiment of the present invention comprises four molecules: the first is a Chemiluminescent Molecule, the second is a Chemiluminescent Substrate, the third is an Intercalating Dye and the fourth is Nucleic Acids. The absorption spectrum of the Intercalating Dye overlaps with the emission spectrum of the Chemiluminescent Molecule.
In one embodiment, the Chemiluminescent Molecule is linked, covalently or noncovalently, to a single strand nucleic acid complementary to the target sequence; this will be called the "Probe". When the "Sample" nucleic acid is denatured and allowed to reanneal in the presence of the "Probe", the "Probe" and the target sequences in the Sample will form double stranded DNA. This double stranded DNA will in turn associate with the Intercalating Dye. The intercalation of the Intercalating Dye into double stranded will shift the absorption spectrum of the Intercalating Dye to overlap with the emission spectrum of the Chemiluminescent Molecule.
Finally, when the Chemiluminescent Molecule is provided with Chemiluminescent Substrate, it will generate the energy to excite the Intercalated Dye molecules and in turn cause them to emit photons at their emission wavelengths. These photons can be detected/counted. One method to quantitate the light emitted by the dye is to apply a filter that is able to discriminate between light emitted at the lower wavelength from light emitted by the intercalated dye. The efficiency with which energy produced by the Chemiluminescent Molecule is captured by the intercalated Intercalating Dye molecules will depend on the distance between them. The light emitted by the Intercalating Dye is a function the distance between the light source (Chemiluminescent Molecule) and the Intercalating Dye. If Excitation by the Chemiluminescent Molecule occurs by Resonance Energy Transfer then Forster's Equation applies. Forster's Equation
states that the transfer of excitation energy between the donor (Chemiluminescent Molecule such as luciferase) and acceptor (Intercalating Dye such as PICOGREEN®) drops off as the 6fll power of the distance between the two.
An advantage of the present invention is that no light source aside from the Chemiluminescent Molecule is necessary for detection. Further, the association of the Chemiluminescent Probe with double strand DNA can be measured without physical separation of the target from other double strand nucleic acid, as only double strand DNA with intercalated Intercalating Dye by close physical association with the Chemiluminescent Probe will produce signal over background. This aspect of the invention alleviates the need for washes, a significant advantage as would be recognized by one with skill in the art. Any detector that can discriminate between the shorter and longer spectra wavelengths can be utilized in this assay system. These include, but are not limited to luminometers, fluorimeters, and CCD cameras equipped with a filter to remove shorter wavelengths in the range of that emitted by the Chemiluminescent Molecule.
C. Embodiment having Probe directly labeled with Chemiluminescent Molecule and Sample in solution
Figure 1 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence in solution phase. Here the Chemiluminescent Molecule is a luciferase. The luciferase is covalently attached to a ssDNA probe. This Bioluminescent Probe is added to a sample containing target sequence nucleic acid in solution and a nucleic acid stain. Coelenterazine is then added to activate the luciferase. The luciferase excites the nucleic acid intercalated stain that in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
D. Embodiment having Probe directly labeled with Chemiluminescent Molecule and Sample is immobilized on Solid Support
Figure 2 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence immobilized on a solid support. Here the Chemiluminescent Molecule is a luciferase. The luciferase is covalently attached to a ssDNA probe. This Bioluminescent Probe is then added to the Sample with target nucleic acid immobilized in a solid support. Nucleic acid stain is present in solution in the
Sample. Coelenterazine is then added to activate the luciferase. The luciferase excites the nucleic acid intercalated stain that in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
E. Embodiment having Probe indirectly labeled with Chemiluminescent Molecule and Sample in solution
Figure 3 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic of specific sequence in solution phase. Here the Chemiluminescent Molecule is a luciferase. The luciferase is noncovalently conjugated to a biotinylated ssDNA probe through a streptavidin intermediate. Because a single streptavidin molecule may bind four biotin molecules, biotinylated probe DNA, biotinylated luciferase and streptavidin may be mixed in the appropriate ratios to generate a Bioluminescent Probe. The Bioluminescent Probe is added to a sample containing target sequence nucleic acid and a nucleic acid stain. Coelenterazine is added to activate the luciferase. The luciferase then excites the nucleic acid intercalated stain, which in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
F. Embodiment having Probe indirectly labeled with Chemiluminescent Molecule and Sample is immobilized on Solid Support
Figure 4 is a schematic representation of a Chemiluminescent Probe being used to quantitate nucleic acid of specific sequence immobilized on a solid support. Here the
Chemiluminescent Molecule is a luciferase. The luciferase is noncovalently conjugated to
a biotinylated ssDNA probe through a streptavidin intermediate. Because a single streptavidin molecule may bind four biotin molecules biotinylated probe DNA, biotinylated luciferase and streptavidin may be mixed in the appropriate ratios to generate a Bioluminescent Probe. This Bioluminescent Probe is then added to the Sample with target nucleic acid immobilized in a solid support. Nucleic acid stain is present in solution in the Sample. Coelenterazine is then added to activate the luciferase. The luciferase excites the nucleic acid intercalated stain, which in turn emits a spectrum of light with a maximal wavelength of 520 nm. Light with wavelengths below 500 nm is filtered out. Light with wavelengths greater than 500 nm is permitted to pass to a detector.
G. Embodiment having Probe directly labeled with Chemiluminescent Molecule, Probe immobilized on Solid Support and Sample in solution
H. Uses of the Invention
The invention is a general method for detecting and quantitating target nucleic acid sequences in a heterogeneous mixture of nucleic acids. The detection of nucleic acids is important for many applications, including (but not limited to) diagnostic measurements of nucleic acids in bodily tissues and fluids as would be readily understood by one of skill in the art.
The method serves to monitor the increase or decrease of stainable nuclei acid that is contacted to a Chemiluminescent Molecule. Stainable nucleic acid is any polymer of nucleic acid into which Intercalating Dyes will incorporate as opposed to other biological molecules. Upon binding, these Intercalating Dyes undergo a change in their electronic configuration that makes them fluoresce in the presence of the appropriate excitation wavelength.
This will occur when the mass of stainable nucleic acid that is contacted to the Chemiluminescent molecule is altered. This includes but is not limited to the following:
a. The method measures enzymatic activity that polymerizes the extension of a nascent strand, through the incorporation of nucleotides or nucleotide analogs, of
nucleic acid when the extended strand or in the case of duplex its complement are contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present. Said activity includes but is not limited to RNA polymerases, DNA polymerases and telomerases. The invention also serves to detect inhibitors of the activities thereof;
b. The method measures enzymatic activity that degrades nucleic acid when the nucleic acid is contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present. Said activity includes but is not limited to RNA exonucleases, RNA endonucleases, DNA exonucleases and DNA endonucleases.
The invention also serves to detect inhibitors of the activities thereof;
c. The method measures enzymatic activity that facilitates the attachment or ligation of separate nucleic acid molecules when one of the nucleic acid molecules is contacted to a Chemiluminescent Molecule and a light responsive intercalating dye is present. Said activity includes but is not limited to DNA ligases. The invention also serves to detect inhibitors of the activities thereof;
d. The method measures enzymatic activity that facilitates the recombination of nucleic acid duplex molecules when one of the nucleic acid duplex molecules is contacted to the Chemiluminescent Molecule, a light responsive intercalating dye is present and the mass of the nucleic acid duplex of the recombined product is different than the mass of the nucleic acid duplex of the non-recombined molecule. Said activity includes but is not limited to recombinases and integrases. The invention also serves to detect inhibitors of the activities thereof;
e. The method measures enzymatic activity that facilitates the attachment or ligation of duplex nucleic acid molecules to protein molecules when the protein molecules are contacted to or are a Chemiluminescent Molecule and a light responsive intercalating dye is present. The invention also serves to detect inhibitors of the activities thereof.
All patents and publications referred to herein are expressly incorporated by reference in their entirety. The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
EXAMPLES
Example One
Luciferase activation of DNA intercalated dye is proportional to the DNA present. Objective:
The objective of this experiment was to determine if the luciferase enzyme of Gaussia princeps (glue) is sufficient to act as an excitation source for a fluorophore that is staining double stranded (ds) DNA. Specifically, the experiment is intended to determine if the glue which emits light at apeak of 480 nm can excite a dsDNA intercalated nucleic acid stain with an excitation maximum in the range of 495 nm to 500 nm and an emission maximum of approximately 520 nm. This would be done by detecting light from a glue, fluorescing nucleic acid stain/ dsDNA mixture which has had wavelengths below 500 nm filtered out.
Materials and Methods:
The Gaussia princeps luciferase was from Avidity LLC (Denver, CO). The SYBR Green I nucleic acid stain and the linearized dsDNA ladder were obtained from Invitrogen (Carlsbad, CA).
The reactions for the detection of dsDNA were as follows. Dilutions of dsDNA, SYBR Green I and glue were made with 50 mM Tris- HCl pH 7.8, 600 mM NaCl, 1 mM EDTA and 20% BPER II (Pierce Biotechnology, Rockford, IL). Coelenterazine (Nanolight International, Pinetop, CA) was diluted into PBS, 1 mM EDTA. The final concentration of coelenterazine in the reaction was 50 uM.
Various amounts of dsDNA were preincubated with 0.78 μl of 200 x concentrated SYBR Green I in a volume of 50 μl. A Ix concentration is relative and is that defined by the manufacturer as the standard assay amount for DNA detection. Glue
(50ng) in 5 μl was added to the prestained dsDNA. The luciferase reaction was initiated by the addition of 100 μl of coelenterazine. The reactions were performed in the wells of a white polystyrene 96 multiwell plate (Evergreen Scientific, Los Angeles, CA). Light emitted by the reaction was detected with a CCD camera (Raytest, Straubenhardt, Germany). Quantitative analysis of the images obtained with camera was performed with the AIDA software package (Raytest) that was included with the camera. The capture of light from the reaction by the camera began 10 sec after the reaction was initiated. Light was in 1, 15 min increment (Figure 5). Discrimination between light emitted by the glue and that emitted by the excited nucleic acid stain/dsDNA duplex was made through the insertion of a filter between the light producing reaction and the aperture of the CCD camera. The aperture setting for the camera was either 0.95 or 11. The filter (Clare Chemical Research, Dolores, CO) has been demonstrated to effectively image fluorescing nucleic acid stains that are excited by light wavelengths in the 400 nmto 500 nm range and emit at wavelengths higher than 500 nm when complexed with dsDNA. It does so by significantly filtering out wavelengths lower than 500 nm.
Results:
It was determined that under the conditions used, 50 ng of glue generated light levels that were within the dynamic range of detection for the CCD camera both with and without the filter. It was also determined that the light generated by this amount of enzyme was as expected, significantly reduced when the filter was used. This level of enzyme was then assayed in the presence of different amounts of dsDNA (Figure 5).
Figure 5 depicts the light generating reaction performed with 50 ng of glue and coelenterazine in the presence of SYBR Green I and 0 μg, 0.063 μg, 0.125 μg, 0.25 μg, 0.5 μg, 1 μg, and 2 μg of linearized dsDNA (spots 1-7 respectively). Part A shows the
CCD camera images for the reactions with filter. Part B shows the same data as relative intensity per spot.
In this experiment it was clearly shown that when both the luciferase enzyme and nucleic acid stain were held constant and the amount of dsDNA was increased, the light
produced at longer wavelengths increased proportionally. This demonstrates the ability of glue to act as an intrinsic light source to activate nucleic acid intercalated dye.
Example 2 Luciferase activation of a DNA intercalated dye is proportional to the amount of dye present.
Objective:
The objective of this experiment was to determine if the luciferase enzyme of Gaussia princeps (glue) is sufficient to act as an excitation source for a fluorophore that is staining double stranded (ds) DNA. Specifically, the experiment is intended to determine if the glue which emits light at a peak of 480 nm can excite a dsDNA intercalated nucleic acid stain with an excitation maximum in the range of 495 nm to 500 nm and an emission maximum of approximately 520 nm. This would be done by detecting light from a glue, fluorescing nucleic acid stain/ dsDNA mixture which has had wavelengths below 500 nm filtered out.
Materials and Methods:
The reactions were performed with the same reagents and under the same conditions as described in Example 1. However, in this experiment the concentrations of dsDNA and glue are held constant and the concentration of SYBR Green I is varied (Figure 6). dsDNA (2ug) was preincubated with SYBR Green I in a volume of 50 μl to final concentrations of Ox, 0.16x, 0.3x, 0.63x, 1.3x, 2.5x, 5x, and 10x. Glue (50ng) in 5 μl was added to the prestained dsDNA. The luciferase reaction was initiated by the addition of 100 μl coelenterazine.
The amount of light emitted over 500 nm in each reaction was determined as described in Example 1. The same reactions were also performed in the absence of dsDNA (-dsDNA).
Results:
It was determined that under the conditions used, 50 ng of glue generated light levels that were within the dynamic range of detection for the CCD camera both with and without the filter. It was also determined that the light generated by this amount of enzyme was as expected, significantly reduced when the filter was used. This level of enzyme was then assayed in the presence of different amounts of SYBR Green I (Figure 6). Figure 6 depicts the light generating reaction performed with 50 ng of glue and coelenterazine in the presence (+ dsDNA) or absence (-dsDNA) of linearized dsDNA and SYBR Green I to final concentrations of Ox, 0.16x, 0.3x, 0.63x, 1.3x, 2.5x, 5x, and 1Ox (spots 1-8 respectively). Part A shows the CCD camera images for the reactions with filter. Part B shows the same data as relative intensity per spot.
In this experiment it was clearly shown that when both the luciferase enzyme and dsDNA were held constant and the amount of SYBR Green I was increased, the light produced at longer wavelengths increased proportionally. This demonstrates the ability of glue to act as an intrinsic light source to activate dsDNA duplex intercalated fluorescing dye.
Example 3
Proximity dependent activation of a dsDNA intercalated dye.
Objective The purpose of this experiment was to demonstrate the dependence that proximity of the Chemiluminescent Molecule to the nucleic acid intercalated dye has on activation of the dye. In this experiment biotinylated dsDNA target is mixed with biotinylated glue. In this mixture there is no association of the two species of molecules with one another. Upon addition of increasing amounts of streptavidin the dsDNA and glue become associated with one another with the streptavidin acting as an intermediate (Figure 7A). This is due to the tight non-covalent binding of the biotin moieties on the dsDNA and glue to the four available biotin-binding sites on the streptavidin. As the amount of streptavidin increases the greater the number of molecules of dsDNA that are placed in close proximity to the glue molecules also increases. If the activation of the intercalated dye is dependent on the close proximity of the glue to the dye then the amount of
fluorescence at longer wavelengths should increase as the amount of streptavidin increases.
Materials and Methods
The reactions were performed with the same reagents and under the same conditions as described in Example 1. However, in this experiment the dsDNA used was made by annealing two complementary synthetic (Sigma-Genosys, The Woodlands, TX) oligonucleotides of DNA 85 and 95 nucleotides in length. One of the oligomers (95 nucleotides) was biotinylated at the 5' end. Streptavidin was from Pierce Biotechnology (Rockford, IL). dsDNA (2.5 pmole of biotinylated 5' end per reaction) was preincubated with
0.78 μl of 20Ox SYBR Green I in a volume of 50 μl. Glue (50ng) in 5 μl was added to the prestaήied dsDNA. Streptavidin in various amounts in 5 μl ddH20 was added to this mix. The mix was incubated 15 min with gentle shaking at room temperature. The luciferase reaction was initiated by the addition of 100 μl of coelenterazine. The amount of light emitted over 500 nm in each reaction was determined as described in Example 1 (Figure 7B, Figure 7C). Results
Figure 7 depicts the light generating reaction performed with 50 ng of biotinylated glue and coelenterazine in the presence of PicoGreen dye. The DNA target for each reaction was at concentration of 2.5 pmole per biotinylated end per reaction. Streptavidin was present at 0.013 pmole, 0.026 pmole, 0.05 pmole, 0.1 pmole, 0.2 pmole, 0.42, 0.84 pmole (spots 1-8 respectively). Part A shows a schematic diagram of the experimental design. Part B shows the CCD camera images for the reactions with filter. Part C shows the CCD camera images for each reaction assessed as relative intensity per spot. In this experiment it was shown that the amount of light that can pass through the filter to the CCD camera is directly proportional to the amount of streptavidin that is added. All other components, luciferase, intercalating dye, dsDNA, and coelenterazine are the same in each reaction. The streptavidin serves to bring the Chemiluminescent Molecule (glue), and stained nucleic acid into a single complex in close proximity to one another. As more streptavidin is added more of the complex is created. The increase in complex is directly proportional the amount of longer wavelength light.
Example 4
Figure 8 depicts a hypothetical experiment representing a further application of the method. Each data point represents the intensity of a light emitting reaction with the amount of single stranded DNA target increasing in each reaction going from left to right. Gluc/DNA probe, PICOGREEN® and coelenterazine are held constant. Reactions are with either a) Probe with sequence complementary to the target DNA or b) Probe with sequence not complementary to the target DNA.
Claims
1. A method comprising: a) providing a sample suspected of containing a nucleic acid of interest, b) contacting the sample with a nucleic acid molecule complementary to the nucleic acid of interest to form a nucleic acid duplex; c) contacting the sample with an intercalating dye to generate a dye-bound nucleic acid duplex, and a chemiluminescent molecule; d) activating said chemiluminescent molecule to produce light, wherein the light excites the intercalating dye in the dye-bound duplex, and e) detecting the light emitted by the intercalating dye, whereby a nucleic acid of interest is detected.
2. The method of claim 1 wherein said nucleic acid of interest is associated with said chemiluminescent molecule.
3. The method of claim 1 wherein said nucleic acid complementary to the nucleic acid of interest is associated with said chemiluminescent molecule.
4. The method according to claim 1, wherein said nucleic acid of interest is selected from the group consisting of DNA, RNA, and derivative thereof.
5. The method according to claim 1 , wherein the nucleic acid complementary to the nucleic acid of interest is selected from the group consisting of DNA, RNA, and derivative thereof.
6. The method according to claim 1, wherein said chemiluminescent molecule is activated by a luciferin.
7. The method of claim 6, wherein said lucifeiϊn is selected from the group consisting of firefly luciferin, coelenterazine, bacterial luciferin, dino flagellate luciferin, vargulin, and synthetic analogs of the foregoing that are oxidized in the presence of a luciferase in a reaction the produces bioluminescence.
8. The method according to claim 1, wherein at least one of the nucleic acids of interest and the complementary nucleic acid is bound to a solid support.
9. The method according to claim 1, wherein the intercalating dye is selected from the group consisting of PICOGREEN® and SYBR GREEN I®.
10. The method according to claim 1, wherein detecting the light is qualitative.
11. The method according to claim 1 , wherein detecting the light is quantitative.
12. The method according to claim 1, wherein said chemiluminescent molecule is a luciferase.
13. The method of claim 12, wherein the luciferase is a luciferase from a system selected from the group consisting of Renilla, Gaussia, Pleuromamma, Aequorea, Obelia, Porichthys, Aristostomias, Odontosyllis, Oplophorus, firefly, bacterial, Cavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus, Chiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia; cuttlefish, Sepiolina, Oplophorus, Sergestes, Gnathophausia; Argyropelecus, Yarella, Diaphus, and Neoscopelus systems.
14. The method of claim 13, wherein the luciferase is Gaussia princeps luciferase.
15. The method of claim 3, wherein said chemiluminescent molecule is associated with said complementary nucleic acid molecule by means selected from the group consisting of covalent and non-covalent interactions.
16. The method of claim 2, wherein said chemiluminescent compound is associated with said nucleic acid of interest by means selected from the group consisting of covalent and non-covalent interactions.
17. A method comprising: a) providing a sample suspected of containing a nucleic acid of interest, b) contacting the sample with a nucleic acid molecule complementary to the nucleic acid of interest to form a nucleic acid duplex, wherein the nucleic acid incorporates fluorescently labeled nucleotides or nucleotide analogs that fluoresce; c) contacting the sample with a chemiluminescent molecule; d) activating the chemiluminescent molecule to produce light, wherein the light excites the fluorescently labeled nucleotides or nucleotide analogs that fluoresce; and e) detecting the light emitted by the fluorescently labeled nucleotides or nucleotide analogs that fluoresce, whereby a nucleic acid of interest is detected.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT06784583T ATE524564T1 (en) | 2005-06-03 | 2006-06-05 | CHEMILUMINESCENCE NUCLEIC ACID TEST |
DK06784583.4T DK1888788T3 (en) | 2005-06-03 | 2006-06-05 | Chemiluminescence nucleic acid assay |
EP06784583A EP1888788B1 (en) | 2005-06-03 | 2006-06-05 | Chemiluminescence proximity nucleic acid assay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US68764705P | 2005-06-03 | 2005-06-03 | |
US60/687,647 | 2005-06-03 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2006133054A2 true WO2006133054A2 (en) | 2006-12-14 |
WO2006133054A3 WO2006133054A3 (en) | 2007-07-19 |
Family
ID=37498980
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2006/021675 WO2006133054A2 (en) | 2005-06-03 | 2006-06-05 | Chemiluminescence proximity nucleic acid assay |
Country Status (6)
Country | Link |
---|---|
US (2) | US8329397B2 (en) |
EP (1) | EP1888788B1 (en) |
AT (1) | ATE524564T1 (en) |
DK (1) | DK1888788T3 (en) |
ES (1) | ES2371664T3 (en) |
WO (1) | WO2006133054A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2227561A2 (en) * | 2007-12-04 | 2010-09-15 | Panagene, Inc. | Method for selective labeling and detection of target nucleic acids using immobilized peptide nucleic acid probes |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080108097A1 (en) * | 2006-07-24 | 2008-05-08 | National Institute Of Advanced Industrial Science And Technology | Method for producing complex of biotin-labeled cypridina (cypridina noctiluca) luciferase with streptoavidin and method for stabilizing the same |
EP3759118A4 (en) * | 2018-02-20 | 2022-03-23 | William Marsh Rice University | Systems and methods for allele enrichment using multiplexed blocker displacement amplification |
WO2021092795A1 (en) * | 2019-11-13 | 2021-05-20 | 李峰 | Nucleic acid detection method and device |
Family Cites Families (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4678636A (en) * | 1982-09-24 | 1987-07-07 | Gte Products Corporation | Ductile brazing alloy containing reactive metals and precious metals |
US4687636A (en) | 1984-01-03 | 1987-08-18 | Hiram Hart | Separative scintillation proximity assay |
US4794073A (en) * | 1985-07-10 | 1988-12-27 | Molecular Diagnostics, Inc. | Detection of nucleic acid hybrids by prolonged chemiluminescence |
US5340716A (en) * | 1991-06-20 | 1994-08-23 | Snytex (U.S.A.) Inc. | Assay method utilizing photoactivated chemiluminescent label |
US5503985A (en) * | 1993-02-18 | 1996-04-02 | Cathey; Cheryl A. | Disposable device for diagnostic assays |
US5863726A (en) * | 1993-11-12 | 1999-01-26 | Geron Corporation | Telomerase activity assays |
US5804380A (en) * | 1993-11-12 | 1998-09-08 | Geron Corporation | Telomerase activity assays |
GB9417593D0 (en) * | 1994-09-01 | 1994-10-19 | Secr Defence | Luciferase labelling method |
US5861318A (en) * | 1994-11-16 | 1999-01-19 | Pharmacia & Upjohn Company | Scintillation proximity assay for N-acetylgalactosaminyltransferase activity |
US5876995A (en) * | 1996-02-06 | 1999-03-02 | Bryan; Bruce | Bioluminescent novelty items |
DE69938293T2 (en) * | 1998-03-27 | 2009-03-12 | Bruce J. Beverly Hills Bryan | LUCIFERASE, GFP FLUORESCENCE PROTEINS, CODING NUCLEIC CAUSE, AND ITS USE IN DIAGNOSIS |
GB9811483D0 (en) * | 1998-05-29 | 1998-07-29 | Photonic Research Systems Limi | Luminescence assay using cyclical excitation wavelength sequence |
US6465182B1 (en) * | 1999-04-29 | 2002-10-15 | The Regents Of The University Of California | Comparative fluorescence hybridization to oligonucleotide microarrays |
US6448004B1 (en) * | 1999-10-05 | 2002-09-10 | Aventis Pharmaceuticals Inc. | Electrochemiluminescence helicase assay |
AU2137101A (en) * | 1999-12-22 | 2001-07-03 | Biosignal Packard Inc. | A bioluminescence resonance energy transfer (bret) fusion molecule and method ofuse |
US20030203404A1 (en) * | 1999-12-22 | 2003-10-30 | Erik Joly | Bioluminescence resonance energy transfer( bret) system with broad spectral resolution between donor and acceptor emission wavelengths and its use |
US6653079B2 (en) * | 2000-03-13 | 2003-11-25 | Freshgene, Inc. | Methods for detection of differences in nucleic acids |
JP3425623B2 (en) * | 2000-04-03 | 2003-07-14 | 東京工業大学長 | DNA fluorescently labeled probe, fluorescently labeled plasmid |
WO2002035260A2 (en) * | 2000-10-27 | 2002-05-02 | Molecular Devices Corporation | Light detection device |
US6544746B2 (en) * | 2001-08-13 | 2003-04-08 | St. Louis University | Rapid and sensitive proximity-based assay for the detection and quantification of DNA binding proteins |
JP4457001B2 (en) * | 2002-05-31 | 2010-04-28 | セクレタリー・デパートメント・オブ・アトミック・エナジー | MET / FRET based method for target nucleic acid detection in which donor / acceptor moieties are on complementary strands |
US7319041B2 (en) * | 2002-09-27 | 2008-01-15 | Siemens Medical Solutions Diagnostic | Applications of acridinium compounds and derivatives in homogeneous assays |
US20060099646A1 (en) * | 2002-10-11 | 2006-05-11 | Anders Heding | Bret assay |
CN101124472A (en) * | 2004-03-17 | 2008-02-13 | 夏威夷大学 | Sensor constructs and detection methods |
-
2006
- 2006-06-05 EP EP06784583A patent/EP1888788B1/en active Active
- 2006-06-05 DK DK06784583.4T patent/DK1888788T3/en active
- 2006-06-05 AT AT06784583T patent/ATE524564T1/en active
- 2006-06-05 ES ES06784583T patent/ES2371664T3/en active Active
- 2006-06-05 WO PCT/US2006/021675 patent/WO2006133054A2/en active Application Filing
- 2006-06-05 US US11/448,167 patent/US8329397B2/en active Active
-
2012
- 2012-12-11 US US13/711,525 patent/US8735062B2/en active Active
Non-Patent Citations (2)
Title |
---|
ALBA F.J., LUMINESCENCE, vol. 16, no. 3, 2001, pages 247 - 249 |
ALBA F.J.; DABAN J.-R., PHOTOCHEMISTRY AND PHOTOBIOLOGY, vol. 69, no. 4, 1999, pages 405 - 409 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2227561A2 (en) * | 2007-12-04 | 2010-09-15 | Panagene, Inc. | Method for selective labeling and detection of target nucleic acids using immobilized peptide nucleic acid probes |
EP2227561A4 (en) * | 2007-12-04 | 2012-05-16 | Panagene Inc | Method for selective labeling and detection of target nucleic acids using immobilized peptide nucleic acid probes |
Also Published As
Publication number | Publication date |
---|---|
ES2371664T3 (en) | 2012-01-09 |
ATE524564T1 (en) | 2011-09-15 |
EP1888788A4 (en) | 2009-04-01 |
EP1888788A2 (en) | 2008-02-20 |
US8735062B2 (en) | 2014-05-27 |
WO2006133054A3 (en) | 2007-07-19 |
EP1888788B1 (en) | 2011-09-14 |
US8329397B2 (en) | 2012-12-11 |
DK1888788T3 (en) | 2011-11-28 |
US20130323724A1 (en) | 2013-12-05 |
US20100221703A1 (en) | 2010-09-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11001877B2 (en) | Systems and methods for multiplex analysis of PCR in real time | |
US7141370B2 (en) | Bioluminescence regenerative cycle (BRC) for nucleic acid quantification | |
WO2005059548A1 (en) | Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor | |
US20140378327A1 (en) | Real-time multiplexed hydrolysis probe assay using spectrally identifiable microspheres | |
US8735062B2 (en) | Chemiluminescence proximity nucleic acid assay | |
JP2004532044A5 (en) | ||
Liu et al. | Recent advances in the exonuclease III-assisted target signal amplification strategy for nucleic acid detection | |
Strohsahl et al. | Towards single-spot multianalyte molecular beacon biosensors | |
US20220136040A1 (en) | Using tethered enzymes to detect nucleic acids | |
Mainguy | A DNAZYME-LINKED SIGNAL AMPLIFICATION ASSAY FOR BACTERIAL BIOSENSING |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2006784583 Country of ref document: EP |